Hot-water extraction, ethanol precipitation, and silkworm bioassays detect innate immune-stimulating activity in coffee extracts

A workflow combining hot-water extraction and ethanol precipitation can provide macromolecule-enriched fractions, but rapid in vivo evaluation of their innate immune-stimulating activity is not well established. We used coffee as a test matrix and evaluated the resulting fractions using a silkworm muscle-contraction assay and a Pseudomonas aeruginosa infection assay. Ethanol-precipitated fractions were prepared from three coffee origins, with or without lactic acid bacterial fermentation, under light- or dark-roast conditions. All sample groups showed measurable muscle-contraction activity. Specific activity ranged from 0.74 to 9.1 U/mg and was higher after light roasting in 11 of 12 matched conditions. Selected fractions increased survival at approximately 24 h after P. aeruginosa challenge, most clearly among Colombia EX samples; protection varied with bean origin, fermentation condition, roast, and dose. Non-fermented fractions were also active, indicating that fermentation was not uniformly required. These results support the feasibility of combining hot-water extraction, ethanol precipitation, and silkworm testing to detect bioactivity in macromolecule-enriched fractions. The method may be useful for prioritizing samples for chemical characterization.